PF4-dependent P-selectin expression assay in comparison to the heparin-induced platelet activation assay for the diagnosis of heparin-induced thrombocytopenia

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a severe complication characterized by thrombocytopenia and thrombosis. The presence of antibodies against heparin/platelet factor 4 (PF4) complexes is a key indicator for HIT. Diagnostic laboratory testing generally includes detection of anti-heparin/PF4 and/or functional testing such as the heparin-induced platelet activation assay (HIPAA). However, current functional tests are time-consuming and require specific technical skills. The PF4-dependent P-selectin expression assay (PEA) is a flow cytometry-based test that evaluates activation of donor platelets in the presence of HIT patient serum. METHODS: We conducted the PEA with 23 patient sera that were positive in both the anti-PF4/heparin enzyme-linked immunosorbent assay (ELISA) and HIPAA and 26 sera that tested negative in the anti-PF4/heparin ELISA. We next compared the PEA to the HIPAA using a retrospective clinical cohort of 195 sera of suspected HIT patients and tested the reproducibility of the PEA. RESULTS: The PEA was found to have a sensitivity of 73.9% and a specificity of 73.1% compared to the HIPAA. In our retrospective clinical cohort, we found that 74.4% (145 out of 195 samples) had identical results in both the HIPAA and PEA, with a specificity of 64.94% and sensitivity of 80.51% for the PEA compared to the HIPAA. Reproducibility testing of the PEA across three independent runs demonstrated consistent results in 83.3% (30 out of 36 samples). DISCUSSION: These findings suggest that the PEA is a promising functional test for HIT laboratory diagnostics based on the relatively high sensitivity; however, further studies are needed to validate its clinical applicability.

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In 195 sera from suspected HIT patients, a flow-cytometric PF4-dependent P-selectin expression assay agreed with the heparin-induced platelet activation assay in 74.4% of samples, showing 80.51% sensitivity and 64.94% specificity, with consistent results in 83.3% of reproducibility runs.

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